The biosynthesis of the myelin proteolipid will be investigated using specific antibody to identify the proteolipid. The use of the electroblot procedure will allow us to immunologically identify proteolipid and DM 20 during in vivo development. The unique capabilities of the electroblot method will also permit identification of any other crossreacting proteins in brain which have different electrophoretic mobilities on gels. Amino acid and fatty acid incorporation into the proteolipid and DM 20 will be compared in rat brain slices to assess possible biosynthetic relationships between the proteins. Pulse and pulse-chase studies are designed to separate the synthesis (amino acid incorporation) and processing (fatty acid incorporation) steps either temporally or with protein synthesis or Golgi processing inhibitors. Samples will be analyzed by both immunoprecipitation and chloroform-methanol solubilization to identify proteolipid, DM 20 and any other crossreacting proteins. Studies of amino acid and fatty acid incorporation into proteolipid and DM 20 in dysmyelinating mouse mutants will investigate genetically induced changes in the synthesis or processing of proteolipid. To further define the different synthesis and processing steps, amino acid and fatty acid incorporation into proteolipid in a cell-free protein synthesis system will be studied. After initial translated products of free and membrane-bound rat brain polysomes are investigated, proteolipid processing will be studied by adding rat brain smooth endoplasmic reticulum to the incubation mixture. These studies will provide information on proteolipid biosynthesis which can only be obtained using proteolipid antibody. This information is important for our understanding of the synthesis and assembly of CNS myelin and ultimately for our understanding of myelin diseases such as multiple sclerosis or metachromatic leukodystrophy.